molecules Article
A New Alkamide with an Endoperoxide Structure from Acmella ciliata (Asteraceae) and Its in Vitro Antiplasmodial Activity Narjara Silveira 1 , Julia Saar 1,2 , Alan Diego C. Santos 3 , Andersson Barison 3 , Louis P. Sandjo 1 , Marcel Kaiser 4,5 , Thomas J. Schmidt 2, * and Maique W. Biavatti 1, * 1
2 3 4 5
*
Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Bloco J/K, Universidade Federal de Santa Catarina, Florianópolis 88040-900, SC, Brazil;
[email protected] (N.S.);
[email protected] (J.S.);
[email protected] (L.P.S.) Institut für Pharmazeutische Biologie und Phytochemie (IPBP), University of Münster, PharmaCampus, Corrensstraße 48, Münster D-48149, Germany Departamento de Química, Universidade Federal do Paraná, Curitiba 81530-900, PR, Brazil;
[email protected] (A.D.C.S.);
[email protected] (A.B.) Swiss Tropical and Public Health Institute (Swiss TPH), Socinstr. 57, Basel CH-4051, Switzerland;
[email protected] University of Basel, Petersplatz 1, Basel CH-4003, Switzerland Correspondence:
[email protected] (T.J.S.);
[email protected] (M.W.B.); Tel.: +49-251-83-33378 (T.J.S.); +55-48-3721-3493 (M.W.B.)
Academic Editor: Dereck J. McPhee Received: 12 May 2016; Accepted: 2 June 2016; Published: 11 June 2016
Abstract: From the aerial parts of Acmella ciliata (H.B.K.) Cassini (basionym Spilanthes ciliata Kunth; Asteraceae), three alkamides were isolated and identified by mass- and NMR spectroscopic methods as (2E,6E,8E)-N-isobutyl-2,6,8-decatrienamide (spilanthol, (1)), N-(2-phenethyl)-2E-en-6,8nonadiynamide (2) and (2E,7Z)-6,9-endoperoxy-N-isobutyl-2,7-decadienamide (3). While 1 and 2 are known alkamides, compound 3 has not been described until now. It was found that the unusual cyclic peroxide 3 exists as a racemate of both enantiomers of each alkamide; the 6,9-cis- as well as the 6,9-trans-configured diastereomers, the former represents the major, the latter the minor constituent of the mixture. In vitro tests for activity against the human pathogenic parasites Trypanosoma brucei rhodesiense and Plasmodium falciparum revealed that 1 and 3 possess activity against the NF54 strain of the latter (IC50 values of 4.5 and 5.1 µM, respectively) while 2 was almost inactive. Compound 3 was also tested against multiresistant P. falciparum K1 and was found to be even more active against this parasite strain (IC50 = 2.1 µM) with considerable selectivity (IC50 against L6 rat skeletal myoblasts = 168 µM). Keywords: Acmella ciliata; jambu; Asteraceae; antiplasmodial activity; alkamide; endoperoxide; Plasmodium falciparum
1. Introduction Acmella ciliata (H.B.K.) Cassini (basionym: Spilanthes ciliata Kunth; Asteraceae) is a herb native to tropical regions of South America [1]. Known under the common name jambu in Brazil, it is used as a spice and in traditional medicine for the treatment of toothaches and is the active ingredient in the herbal medicinal product Spolera® used to treat abrasions and distortions [2]. Previous investigations have been carried out with this species that led to the isolation of several alkylamides (alkamides) [3,4], including the well-known bioactive isobutylamide spilanthol responsible for analgesic [5], anti-inflammatory [6] and antimalarial [7] in vitro activities.
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The closely related species Acmella oleracea (L.) R. K. Jansen (basionym: Spilanthes oleracea; synonym: Spilanthes acmella var. oleracea) is widely used as spice for food and in folk medicine for stomatitis, cold and toothaches and is one of the plants constituting the traditional African antimalarial drug “Malarial-5” [8]. Molecules 2016, 21, 765 2 of 10 Malaria is a disease caused by various Plasmodium species and represents a public health problem causing many hundreds thousands of deaths year, (basionym: especially to people living under poor The closely relatedofspecies Acmella oleracea (L.) R.per K. Jansen Spilanthes oleracea; synonym: Spilanthesand acmella var. oleracea)communities is widely used in as tropical spice for food and in According folk medicinetofor stomatitis, circumstances in vulnerable countries. the latest available and toothaches and is cases one ofoccurred the plants and constituting the traditional African antimalarial data, ancold estimated 214 million the disease killed about 438,000 peopledrug in 2015 [9]. “Malarial-5” [8]. In this way, WHO recommends artemisinin-based combination therapies (ACTs) as the first line Malaria is a disease caused by various Plasmodium species and represents a public health problem treatment of uncomplicated caused by theper P. year, falciparum parasite. However, sincepoor 2006, after causing many hundreds malaria of thousands of deaths especially to people living under the firstcircumstances artemisinin-resistant case was noticedinintropical Cambodia, theAccording resistance to latest artemisinin-derived and in vulnerable communities countries. to the available data,become an estimated 214 million cases occurred and the disease about people in 2015 [9]. drugs has a global concern [10]. Therefore, the killed search for438,000 new antimalarial drugs has In increasing this way, WHO recommends artemisinin-based combination therapies (ACTs) as the alkamides first line received attention, including natural products. In a recent study, several from treatment of uncomplicated malaria caused by the P. falciparum parasite. However, since 2006, after Achillea ptarmica (Asteraceae) were found to be active against Plasmodium falciparum and some other the first artemisinin-resistant case was noticed in Cambodia, the resistance to artemisinin-derived unicellular eukaryotic parasites such as Trypanosoma brucei rhodesiense, responsible for East African drugs has become a global concern [10]. Therefore, the search for new antimalarial drugs has received Humanincreasing Trypanosomiasis Hence, an investigation alkamide-containing for attention, [11]. including natural products. In of a further recent study, several alkamidesAsteraceae from alkamides withptarmica activity against such ofagainst interest. Therefore, in the and present Achillea (Asteraceae) werepathogens found to bewas active Plasmodium falciparum someinvestigation, other unicellular on eukaryotic parasites such asciliata Trypanosoma brucei rhodesiense, East African we concentrated alkamides of Acmella with potential activityresponsible against theformentioned parasites. Human Trypanosomiasis [11]. Hence, an investigation of further alkamide-containing Asteraceae for alkamides with activity against such pathogens was of interest. Therefore, in the present investigation, 2. Results and Discussion we concentrated on alkamides of Acmella ciliata with potential activity against the mentioned parasites.
2.1. Isolation and Characterization of Alkamides 2. Results and Discussion
The hexane fraction of the ethanol extract from the fresh aerial parts of Acmella ciliata afforded, and Characterization of Alkamides besides2.1. theIsolation well-known isobutylamide spilanthol ((2E,6E,8E)-N-isobutyl-2,6,8-decatrienamide, (1) and a further known compound N-(2-phenethyl)-2E-en-6,8-nonadiynamide (2), ciliata one afforded, new alkamide The hexane fraction of the ethanol extract from the fresh aerial parts of Acmella besides the well-known isobutylamide spilanthol ((2E,6E,8E)-N-isobutyl-2,6,8-decatrienamide, (2E,7Z)-6,9-endoperoxy-N-isobutyl-2,7-decadienamide (3) (Figure 1). Their chemical (1) structures and aestablished further known (2), one new were fully oncompound the basesN-(2-phenethyl)-2E-en-6,8-nonadiynamide of 1D and 2D NMR and high-resolution massalkamide spectrometric (2E,7Z)-6,9-endoperoxy-N-isobutyl-2,7-decadienamide (3) (Figure 1). Their chemical structures were (HR-MS) data. fully established on the bases of 1D and 2D NMR and high-resolution mass spectrometric (HR-MS) data. O
O N H
N H
(2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide (spilanthol, 1)
N-(2-phenethy)-2E-en-6,8-nonadiynamide 2 O
O
O
N H
(2E,7Z)-6,9-endoperoxy-N-isobutyl-2,7-decadienamide 3 Figure 1. Structures of the isolated alkamides.
Figure 1. Structures of the isolated alkamides. Compound 1 was obtained as a green oil and identified as spilanthol by comparison of its spectral data with those described in the literature [12]. Compound 1 was obtained as a green oil and identified as spilanthol by comparison of its spectral Compound 2 was obtained as a yellow oil. It was previously isolated from A. ciliata [4] and its data with those described in the literature [12]. chemical structure was confirmed by comparison of the mass and 1H-NMR spectroscopic data with Compound 2 was obtained those reported in the literature.as a yellow oil. It was previously isolated from A. ciliata [4] and its 1 H-NMR spectroscopic data with chemical structure was confirmed by comparison the+ESI mass andmass Compound 3 was also obtained as a yellow oil.ofThe QqTOF spectrum obtained under UHPLC coupling allowed establishment of the elemental formula as C14H23NO3 ([M + H]+ at m/z those reported in the literature. 254.1759, [M3 +was Na]+also at m/z 276.1574; as calcd. 254.1751oil. andThe 276.1570, While the isolate Compound obtained a yellow +ESIrespectively). QqTOF mass spectrum obtained yielded one sharp peak in the UHPLC/MS chromatogram, it was found to show NMR signals of two +
under UHPLC coupling allowed establishment of the elemental formula as C14 H23 NO3 ([M + H] at
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m/z 254.1759, [M + Na]+ at m/z 276.1574; calcd. 254.1751 and 276.1570, respectively). While the isolate yielded one sharp peak in the UHPLC/MS chromatogram, it was found to show NMR signals of two very similar isomeric forms (ratio of approximately 3:1) in which most signals resonated at very similar or identical shift values with few exceptions. Nevertheless, the chemical shifts in the proton spectrum, even in parts of the spectrum with strong signal overlap, could all be determined with the help of a 2D J-resolved 1 H-NMR experiment and all 1 H/1 H-coupling constants became accessible by selective homonuclear decoupling measurements. The NMR spectra (Tables 1 and 2) showed the presence of an isobutylamide moiety with 1 H signals at δ 3.14 (2H, H-1’), 1.80 (1H, H-2’) and 0.92 (6H, H-3’ and H-4’) and 13 C resonances at δ 46.8 (C-1’), 28.5 (C-2’) and 20.1 (C-3’, C-4’) for the amine component as well as the amide carbonyl at 166.0 ppm (C-1), the latter correlated with the proton signal at δ 3.14 ppm in the HMBC spectrum (key HMBC correlations are summarized in Figure 2). Table 1. 1 H-NMR data (CDCl3 , 600 MHz) for compounds 3a and 3b. 3a
3b
δH , Mult. (J in Hz)
δH , Mult. (J in Hz)
5.82 ddd (15.2, 1.4 and 1.4) 6.81 ddd (15.2, 7.4 and 6.6)
5.82 ddd (15.2, 1.4 and 1.4) 6.81 ddd (15.2, 7.4 and 6.6)
4
2.32 ddddd (14.9, 8.8, 7.4, 7.2 and 1.4) 2.36 ddddd (14.9, 9.1, 6.6, 5.3 and 1.4)
2.32 ddddd (14.9, 8.8, 7.4, 7.2 and 1.4) 2.36 ddddd (14.9, 9.1, 6.6, 5.3 and 1.4)
5
1.69 dddd (14.2, 9.1, 7.2 and 4.0) 1.87 dddd (14.2, 8.9, 8.8 and 5.3)
1.67 dddd (14.2, 9.1, 7.2 and 4.0) 1.87 dddd (14.2, 8.9, 8.8 and 5.3)
6 7 8 9 10 1’ 2’ 3’ 4’
4.38 ddddd (8.9, 4.0, 2.4, 1.3 and 1.2) 5.83 ddd (10.3, 2.4 and 1.5) 5.86 ddd (10.3, 1.7 and 1.2) 4.66 qddd (6.7, 1.7, 1.5 and 1.3) 1.23 d (6.7) 3.14 dd (6.8 and 6.2) 1.80 th (6.8 and 6.7) 0.92 d (6.7) 0.92 d (6.7)
4.59 ddddd (8.9, 4.0, 2.4, 1.3 and 1.2) 5.78 ddd (10.3, 2.4 and 1.5) 5.83 ddd (10.3, 1.7 and 1.2) 4.70 qddd (6.7, 1.7, 1.5 and 1.3) 1.20 d (6.7) 3.14 dd (6.8 and 6.2) 1.80 th (6.8 and 6.7) 0.92 d (6.7) 0.92 d (6.7)
Position 1 2 3
The corrected multiplicities in the 1 H-NMR spectra were established with the aid of FOMSC3 software [13].
Table 2.
13 C-NMR
data (CDCl3 , 150 MHz) for compounds 3a and 3b. 3a
Position
3b
δC
HMBC
δC
HMBC
1 2 3
166.0 124.4 143.3
C-1, C-4 C-1, C-2, C-4, C-5
165.9 124.4 143.2
C-1, C-4 C-1, C-2, C-4, C-5
4
27.9
C-2, C-3, C-5, C-6 C-2, C-3, C-5, C-6
27.5
C-2, C-3, C-5, C-6 C-2, C-3, C-5, C-6
5
31.5
C-3, C-4, C-6, C-7 C-3, C-4, C-6, C-7
31.0
C-3, C-4, C-6, C-7 C-3, C-4, C-6, C-7
6 7 8 9 10 1’ 2’ 3’ 4’
77.2 127.0 129.3 74.3 18.1 46.8 28.5 20.1 20.1
C-4, C-5, C-7, C-8 C-6, C-9 C-6, C-9 C-7, C-8, C-10 C-8, C-9 C-1, C-2’, C-3’, C-4’ C-1’, C-3’, C-4’ C-1’, C-2’, C-4’ C-1’, C-2’, C-3’
76.8 127.2 129.5 74.2 17.7 46.7 28.5 20.1 20.1
C-4, C-5, C-7, C-8 C-6, C-9 C-6, C-9 C-7, C-8, C-10 C-8, C-9 C-1, C-2’, C-3’, C-4’ C-1’, C-3’, C-4’ C-1’, C-2’, C-4’ C-1’, C-2’, C-3’
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Figure 2. Key HMBC correlations for compound 3.
Figure 2. Key HMBC correlations for compound 3. Two proton signals at δ 5.82 (1H, H-2) and 6.81 (1H, H-3) with a coupling constant of 15.2 Hz were attributed to an E-configured double bond. Their chemical shift values as well as the correlations Two proton signals δ 5.82 H-2) and 6.81 (1H,that H-3) a coupling constant observed in the at HSQC and(1H, HMBC experiments showed thiswith double bond is in the direct of 15.2 Hz neighborhood of the amide carbonyl, that itTheir is partchemical of an α,β-unsaturated amide as correlations were attributed to an E-configured double i.e., bond. shift values as group well such as the that also observed in 1 and 2. Further HMBC correlations of the two mentioned olefinic protons were observed in the HSQC and HMBC experiments showed that this double bond is in the direct observed with the methylene carbons at δ 27.9 (C-4) and 31.5 (C-5). The 1H resonances of two protons neighborhood of the amide carbonyl, i.e., that it is part of an α,β-unsaturated at an additional double bond were observed at δ 5.83 (H-7) and 5.86 (H-8), with a coupling amide constant group such of 10.3 Hz, indicating The corresponding olefinic signals were observed at as that also observed in 1 anda Z-configuration. 2. Further HMBC correlations ofcarbon the two mentioned olefinic protons δ 127.0 and 129.3, respectively. Apart from the aforementioned signals, the 1H-NMR spectrum 1 were observed with the methylene carbons at δ 27.9 (C-4) and 31.5 (C-5). The H resonances of two presented signals at δ 4.38 and 4.66 correlated according to the HSQC spectrum with carbon protons at anresonances additional at δspectrum. 5.83 (H-7) 5.86 (H-8), at δ double 77.2 (C-6) bond and 74.3were (C-9) observed in the 13C-NMR Theseand signals clearly showwith the a coupling 3 carbons in the structure. From the HMBC correlations it became clear presence of two oxygenated sp constant of 10.3 Hz, indicating a Z-configuration. The corresponding olefinic carbon signals were both of these carbons are direct neighbors of the Z-configured double bond, the former showing observed at δthat127.0 and 129.3, respectively. Apart from the aforementioned signals, the 1 H-NMR further correlations with the methylene signals of positions 4 and 5, the latter with a methyl group spectrum presented signals at δat4.38 and 4.66 according to the HSQC spectrum with carbon 1H- correlated (position 10) resonating δ 1.23 in the and at 18.1 ppm in the 13C-NMR spectrum. 13 The signals of the protons and carbons especially at this structural fragment (i.e.,signals positionsclearly 6–10) resonances at δ 77.2 (C-6) and 74.3 (C-9) in the C-NMR spectrum. These show the appeared to have shifted between the major and minor components (see below). 3 presence of two oxygenated sp carbons in the structure. From the HMBC correlations it became clear These NMR data were similar to those described for longipenamide B (N-isobutyl-syn-6,9that both of these carbons are direct neighbors the Z-configured double bond, former showing dihydroxy-2E,7E-decadienamide) known fromof Heliopsis longipes [14] which, however, has an the elemental formula of C 23 H 25 NO 3 , i.e., one degree of unsaturation less than the compound described further correlations with the methylene signals of positions 4 and 5, the latter with here. a methyl group E-configured between C-7 and C-8 and free hydroxy 1 H- and double (position 10) Longipenamide resonating atB δpossesses 1.23 inan the at 18.1bond ppm in the 13 C-NMR spectrum. groups at C-6 and C-9. The lack of two hydrogen atoms in comparison with longipenamide B, The signals of the and carbons especially at this structural fragment (i.e., positions 6–10) together withprotons the Z-configuration of the double bond indicated the presence of a cyclic structure in this part of the molecule which thenand either be a dihydrofuran system, the presence appeared to have shifted between thecould major minor components (seerequiring below). of one free hydroxy group, or a dihydro-1,2-dioxine system, i.e., a cyclic peroxide structure. Since no These NMR data were similar to those described for longipenamide B (N-isobutyl-syn-6,9HMBC correlations were observable either in chloroform-d3 or in acetone-d6 between the protons and dihydroxy-2E,7E-decadienamide) from Heliopsis longipes [14] which, however, carbons at positions 6 and 9, known which would be expected in case of a furanoid system, the compoundhas wasan elemental structure of of (2E,7Z)-6,9-endoperoxy-N-isobutyl-2,7-decadienamide. The formula of Cindeed NO3 , the i.e.,depicted one degree unsaturation less than the compound described here. 23 H25assigned observation that especially the NMR signals for the fragment between C-6 and C-10 were shifted Longipenamide B possesses an E-configured double bond between C-7 and C-8 and free hydroxy between the major and minor isomers pointed towards the presence of two stereoisomers, i.e., that groups at C-6one and C-9. The ofother twothe hydrogen atomsform in comparison longipenamide represents the lack cis-, the trans-configured with respect to with the asymmetric carbons, B, together C-6 and C-9. It might expected bond that theindicated cis-orientedthe protons in the former would show an NOE with the Z-configuration of thebedouble presence of a cyclic structure in this part of correlation, as was previously described for a similar endoperoxide from Zanthoxylum [15]. However, the molecule which could then either be a dihydrofuran system, requiring the presence of one free NOEs could not be observed in the 2D NOESY spectrum for either isomer so that the relative hydroxy group, or a dihydro-1,2-dioxine i.e.,Asa mentioned cyclic peroxide Since stereochemistry could not be clarified insystem, this manner. above, the structure. NMR signals of the no HMBC protons around the endoperoxide ring showed significant shift differences in the minor isomer with correlations were observable either in chloroform-d or in acetone-d between the protons and carbons 3 6 H-6 being shifted downfield most prominently for Δδ 0.21 ppm, in comparison to the major isomer. at positions 6 and 9, which would be expected in case of a furanoid system, the compound was Since no directly comparable data exist in literature, molecular modeling using quantum mechanical indeed assigned the depicted of (2E,7Z)-6,9-endoperoxy-N-isobutyl-2,7-decadienamide. The calculations had to be structure applied to assign the relative configuration for the major and minor isomers. To thisespecially end, the NMRthe shiftNMR values were calculated optimized modelsC-6 of theand energetically observation that signals for for thegeometry fragment between C-10 were shifted most favorable conformers of both diastereomers (6R,9R-trans and 6S,9R-cis) of the de-alkylated between the major and minor isomers pointed towards the presence of two stereoisomers, i.e., that one carboxamide part of 3 (the isobutyl moiety was omitted to save computation time). The geometry
represents the cis-, the other the trans-configured form with respect to the asymmetric carbons, C-6 and C-9. It might be expected that the cis-oriented protons in the former would show an NOE correlation, as was previously described for a similar endoperoxide from Zanthoxylum [15]. However, NOEs could not be observed in the 2D NOESY spectrum for either isomer so that the relative stereochemistry could not be clarified in this manner. As mentioned above, the NMR signals of the protons around the endoperoxide ring showed significant shift differences in the minor isomer with H-6 being shifted downfield most prominently for ∆δ 0.21 ppm, in comparison to the major isomer. Since no directly comparable data exist in literature, molecular modeling using quantum mechanical calculations had to be applied to assign the relative configuration for the major and minor isomers. To this end, the NMR shift values were calculated for geometry optimized models of the energetically most favorable conformers of both diastereomers (6R,9R-trans and 6S,9R-cis) of the de-alkylated carboxamide part
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of 3 (the isobutyl moiety was omitted to save computation time). The geometry optimized models were energy minimized with the B3LYP density functional and the 6-31++G(2d,p) basis set. The NMR shifts were calculated using the GIAO method at the same level of theory. As reported in Table 3, the shift differences resulting for the cis- and trans-diastereomers were in good qualitative agreement with the major (3a) and minor isomers (3b), respectively, of compound 3. The formation of 3 (see Figure 3) is most likely to result from addition of a molecule of singlet Molecules 2016, 21, 765 5 of 10 oxygen to the diene moiety of the E,E-isomer of spilanthol (1) in case of the major isomer 3a, and to 1 itself in case ofmodels the minor isomer 3b. Light-induced the optimized were energy minimized with the B3LYP isomerization density functionalof and the6,7-Z-8,9-E 6-31++G(2d,p)diene of 1 basis set. The more NMR shifts were calculated using the method at the sameearlier level of literature theory. to the (energetically favorable) E,E-isomer is GIAO in agreement with reports on As reported in Table 3, the shift differences resulting for the cis- and trans-diastereomers were in peroxidation of unsaturated fatty acids [15]. In order to evaluate whether compound 3 is a genuine good qualitative agreement with the major (3a) and minor isomers (3b), respectively, of compound product of3.the or3)formed during the extraction the fresh aerial parts The plant’s formationmetabolism of 3 (see Figure is most likely to result from additionprocess, of a molecule of singlet (50 g) of A.oxygen ciliatatowere macerated with chloroform (100 (1) mL) for of 2 the h in a flask protected the diene moiety of the E,E-isomer of spilanthol in case major isomer 3a, and to 1from light itself in case the minor isomer 3b.extract Light-induced isomerization of the 6,7-Z-8,9-E diene of 1 tosolution the with aluminum foil.ofThe chloroform was dried in vacuo and its acetonitrile was (energetically more favorable) E,E-isomer is in agreement with earlier literature reports on then subjected to UPLC-ESI-MS analysis. Moreover, fresh flowers and leaves were quickly pounded peroxidation of unsaturated fatty acids [15]. In order to evaluate whether compound 3 is a genuine and the resulting were probedorby ASAP (Atmospheric solid analysis probe) performed product ofjuices the plant’s metabolism formed during the extraction process, the fresh aerial partsMS (50 g) according to established as reported previously, experiments the of A. ciliata were protocols macerated with chloroform (100 mL) fore.g., 2 h in in a[16]. flaskBoth protected from light displayed with aluminumion foil.m/z The 254.1759 chloroformcorresponding extract was driedtoin3vacuo andits its formation acetonitrile solution then already pseudo-molecular so that is likelywas to occur subjected to UPLC-ESI-MS analysis. Moreover, fresh flowers and leaves were quickly pounded and in the living plant to some extent. The structure was further supported by MS2 fragmentation as the resulting juices were probed by ASAP (Atmospheric solid analysis probe) MS performed 1 depicted inaccording Figure to 4. established Taken together, isreported suggested that addition ofBoth O2 experiments proceeds in an uncatalyzed protocolsitas previously, e.g., in [16]. displayed spontaneous This reaction possible by means to of3aso 1,4-cycloaddition sotothat the oxygen the manner. pseudo-molecular ion m/z is 254.1759 corresponding that its formation is[17], likely occur 2 fragmentation already in the living plant to some extent. The structure was further supported by MS is added from either face of the molecules with equal probability (see Figure 3). Therefore, racemates as depicted in Figure 4. Taken together, it is suggested that addition of 1O2 proceeds in an uncatalyzed should result in both cases. Indeed, the isolated mixture 3 was devoid of optical activity (no CD spontaneous manner. This reaction is possible by means of a 1,4-cycloaddition [17], so that the oxygen cotton effects between 450 and 190 so that result is in (see agreement the postulated means is added from either face of thenm) molecules withthis equal probability Figure 3). with Therefore, racemates of formation. It can becases. concluded that 3 is actually a mixture all four possible stereoisomers should resultthus in both Indeed, the isolated mixture 3 was devoid ofof optical activity (no CD cotton effects between 450 and enantiomers 190 nm) so that present this resultas is major in agreement with the postulated means of and the with the 6R,9Sand 6S,9R-cis (3a, approximately 40% each) formation. It can thus be concluded that 3 is actually a mixture of all four possible stereoisomers with 6R,9R- and 6S,9S-enantiomers as the minor constituents (3b, approx. 10% each). Similar alkamides the 6R,9S- and 6S,9R-cis enantiomers present as major (3a, approximately 40% each) and the 6R,9Rwith an endoperoxide bridge as have previously been(3b, described by each). Devkota et alkamides al. [15] aswith constituents of and 6S,9S-enantiomers the minor constituents approx. 10% Similar an a Zanthoxylum species.bridge However, the structure described is a etnew natural product of and endoperoxide have previously been described byhere Devkota al. [15] as constituents a has also Zanthoxylum However, the structure a new naturalFor product and has also not been described asspecies. a synthetic product, to thedescribed best of here our is knowledge. this new compound, we not been described as a synthetic product, to the best of our knowledge. For this new compound, we propose the name dioxacmellamide. propose the name dioxacmellamide.
Figure 3. Hypothetical routes of formation for the stereoisomers of compound 3.
Figure 3. Hypothetical routes of formation for the stereoisomers of compound 3.
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Figure 4. Proposed fragmentation mechanism of the endoperoxide alkamide (m/z 254). Figure 4. Proposed fragmentation mechanism of the endoperoxide alkamide (m/z 254). Table 3. Calculated and experimental chemical shift values and differences for protons at the endoperoxide Table 3. Calculated and experimental chemical shift values and differences for protons at the ring of compound 3. Upfield shifts comparing the first and second columns of calculated and experimental endoperoxide ring of compound 3. Upfield shifts comparing the first and second columns of calculated data, respectively, are colored in blue, downfield shifts in red. and experimental data, respectively, are colored in blue, downfield shifts in red.
Calc. Δ ∆ 6S,9R-cis Calc. 6R,9R-trans 5a 1.426 1.486 Position H6S,9R-cis 6R,9R-trans 5b 2.149 1.510 65a 4.030 4.622 1.426 1.486 5b 2.149 1.510 7 6.098 5.859 4.030 4.622 86 6.032 5.950 7 6.098 5.859 98 4.807 4.849 6.032 5.950 109 0.982 0.998 4.807 4.849
Position H-
10
0.982
0.998
Exp. Δ Δδ Exp Exp. Minor ∆ Major Isomer Isomer 0.059 1.873 1.873 0.000 Major Minor ∆δ Exp ∆δ Calc Isomer Isomer −0.638 1.693 1.669 −0.024 0.591 4.377 4.586 0.209 0.059 1.873 1.873 0.000 ´0.638 1.693 1.669 ´0.024 −0.240 5.830 5.782 −0.048 0.591 4.377 4.586 0.209 −0.082 5.860 5.830 −0.030 ´0.240 5.830 5.782 ´0.048 0.042 4.663 4.696 0.033 ´0.082 5.860 5.830 ´0.030 0.016 1.232 1.203 −0.029 0.042 4.663 4.696 0.033
Δδ Calc
0.016
1.232
1.203
´0.029
2.2. In Vitro Antiplasmodial Activity of Isolated Alkamides 2.2. InAsVitro of Isolated Alkamides partAntiplasmodial of our ongoingActivity efforts to find new compounds with activity against “protozoan” parasites causing neglected wellnew as malaria, the isolated alkamides tested forparasites activity As part of our tropical ongoingdiseases efforts toasfind compounds with activity againstwere “protozoan” against Trypanosoma bruceidiseases rhodesiense (Tbr)as and Plasmodium falciparum (Pf) as were well as for cytotoxicity causing neglected tropical as well malaria, the isolated alkamides tested for activity against Trypanosoma mammalian brucei cells (L6 cell line)(Tbr) usingand established protocols [18].(Pf The IC50 values for rhodesiense Plasmodium falciparum ) asresulting well as for cytotoxicity in vitro mammalian growth inhibition arecell reported in Table 4. Whileprotocols compound displayed only insignificant against cells (L6 line) using established [18].2 The resulting IC50 values for activity againstinhibition either parasite, compounds 1 and 3 werecompound somewhat2more activeonly against Tbr and, in vitro growth are reported in Table 4. While displayed insignificant especially Pf (chloroquine sensitive NF54 strain). In case of 1, antiplasmodial activity at a comparable activity level against strains PFB and K1 of Pf had already been reported [7]. Compound 3, due to its new endoperoxide structure that is related to the most important structure element of the potent
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activity against either parasite, compounds 1 and 3 were somewhat more active against Tbr and, especially Pf (chloroquine sensitive NF54 strain). In case of 1, antiplasmodial activity at a comparable activity level against strains PFB and K1 of Pf had already been reported [7]. Compound 3, due to its new endoperoxide structure that is related to the most important structure element of the potent antimalarial artemisinin, was also tested against the multi-resistant K1 strain of Pf where it displayed even higher activity than against the sensitive strain with an IC50 value of 0.54 µg/mL (2.1 µM) and showed considerable selectivity, i.e., very low cytotoxicity against the L6 cells (S.I. = 78.7). Compound 3 thus appears to be an interesting candidate for in vivo tests against plasmodial infections which are warranted after isolation of larger quantities. Table 4. In vitro antiparasitic activity IC50 values in µg/mL and cytotoxicity (L6 rat skeletal myoblasts) of alkamides 1–3. Mean values are also reported in µM (in brackets) for easier comparison between compounds. Compound
Tbr
Pf (NF54)
Pf (K1)
L6
1 2 3 Pos. contr.
2.88 ˘ 0.06 (13.0) 17.1 ˘ 2.3 (68.1) 5.60 ˘ 0.67 (13.0) 0.004 ˘ 0.001 a
0.99 ˘ 0.12 (4.5) 22.1 ˘ 4.0 (88.0) 1.29 ˘ 0.20 (5.1) 0.002 ˘ 0.001 b
n.t. n.t. 0.54 ˘ 0.14 (2.1) 0.09 ˘ 0.006 b
39.9 ˘ 1.9 (180.3) 60.1 ˘ 9.4 (239.2) 42.6 ˘ 1.7 (168.2) 0.004 ˘ 0.001 c
a
melarsoprol; b chloroquine; c podophyllotoxin.
3. Materials and Methods 3.1. General Procedures Silica gel 60 and Sephadex® LH-20 (GE Healthcare, Uppsala, Sweden) were used for column chromatography, while silica gel 60 F254 aluminum sheets (Silicycle® , Ville de Québec, QC, Canada) were used for analytical thin layer chromatography (TLC). Compounds were visualized by exposure under UV254/365 light and by spraying with p-anisaldehyde reagent followed by heating. All solvents for chromatographic procedures were of analytical grade, while those for MS analysis were of spectrometric grade. NMR experiments were acquired at 303 K in CDCl3 and acetone-d6 on a Bruker (Billerica, MA, USA) AVANCE III NMR spectrometer and on an Agilent (Santa Clara, CA, USA) DD2 600 spectrometer, operating at 14.1 Tesla, observing 1 H and 13 C at 600 and 150 MHz, respectively. High-resolution MS and MS/MS analysis of the pure compounds were performed on a Bruker MicrOTOF-QII ESI-QTOF mass spectrometer coupled to a Dionex (Sunnyvale, CA, USA) Ultimate 3000 RS UHPLC. LC-MS and MS analyses of CHCl3 extract and the plant material were carried on a Waters (Milford, MA, USA) Xevo G2-S QTOF mass spectrometer equipped with exchangeable ESI and ASAP sources and coupled to an Acquity UPLC (Waters) chromatograph. A CD spectrum of 3, recorded in MeOH on a Jasco (Groß-Umstadt, Germany) J815 spectropolarimeter, did not show any cotton effects. 3.2. Botanical Material Aerial parts of Acmella ciliata (H.B.K.) Cassini were collected at the garden of the Associação dos Funcionários Fiscais do Estado de Santa Catarina (AFFESC, Florianópolis, Santa Catarina, Brazil) in October, 2012. The plant material was identified by Dr. Marcos Sobral and a voucher specimen was deposited in the Herbarium of the Botanic Garden of Rio de Janeiro under reference RB 612273. 3.3. Extraction and Isolation A summary of the isolation procedure for compounds 1–3 is shown in Figure 5. Aerial parts of fresh plant (1754 g) were washed with water, air dried and macerated for 3 ˆ 7 days with ethanol 92% (total volume 8 L) at room temperature. The crude extract was filtered and concentrated under reduced
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pressure at 40 ˝ C, yielding 60.9 g of crude ethanol extract. The extract was then dispersed in water and partitioned with solvents of increasing polarity: n-hexane (24.4 g), followed by CH2 Cl2 (0.8 g), and EtOAc (1.6 g), and the residual aqueous fraction was freeze-dried. Molecules 2016, 21, 765 8 of 10
Figure 5. Flow chart for the isolation of the compounds 1–3. Figure 5. Flow chart for the isolation of the compounds 1–3.
The hexane fraction (22.7 g) was initially fractionated by VLC packed with silica gel 60 (particle The hexane (22.7 was initially fractionated by VLC packed with silica gel 60 (particle size size 40–63 µm)fraction (Column A)g) and eluted with gradient solvent systems of n-hexane/acetone in 40–63 µm) (Column and (350 eluted with gradient solvent systems of n-hexane/acetone in increasing increasing polaritiesA) (100:0 mL), 90:10 (300 mL), 80:20 (400 mL), 70:30 (400 mL), 60:40 (600 mL), polarities (350 (200 mL),mL), 90:100:100 (300(300 mL),mL), 80:20 (400 mL), 70:30 (40013mL), 60:40 (600Sub-fraction mL), 40:60 40:60 (300(100:0 mL), 20:80 respectively), yielding sub-fractions. (300 mL), 20:80 (200 mL), 0:100 (300 mL), respectively), yielding 13 sub-fractions. Sub-fraction A4 (4.8 g) was subjected to a silica gel 60 (particle size 40–63 µm) column chromatography (ColumnA4 B) (4.8 was subjected to a silica gelsystems 60 (particle size 40–63 µm) column chromatography (Column B) andg) eluted with gradient solvent of n-hexane/acetone in increasing polarity (100:0 (200 mL), and eluted with 95:05 gradient systems n-hexane/acetone increasing polarity (200 70:30 mL), 98:02 (100 mL), (500solvent mL), 92:08 (300ofmL), 90:10 (100 mL),in85:15 (400 mL), 80:20(100:0 (100 mL), 98:02 (100 mL), 95:05 (500 mL), 92:08 (300 mL), 90:10 (100 mL), 85:15 (400 mL), 80:20 (100 mL), 70:30 (200 mL), 60:40 (100 mL), 0:100 (100 mL), respectively), yielding 48 sub-fractions. The sub-fractions (200 mL),(1.2 60:40 (100 mL), 0:100to (100 mL), silica respectively), yielding sub-fractions. The sub-fractions B18–B27 g) were submitted another gel 60 (particle size48 40–63 µm) column chromatography B18–B27 (1.2 g) were submitted to another silica gel 60 (particle size 40–63 µm) column chromatography (Column C) and eluted with gradient solvent systems of n-hexane/acetone in increasing polarity (Column and eluted withmL), gradient systems n-hexane/acetone increasing polarity (100:0 (50C)mL), 95:05 (100 90:10solvent (50 mL), 80:20of(50 mL), 75:25 (100 in mL), 60:40 (100 mL),(100:0 50:50 (50 80:20 (50 mL), 75:25 (100 18 mL), 60:40 (100 mL), 50:50 (501mL), (50mL), mL),95:05 40:60(100 (100mL), mL),90:10 0:100(50 (50mL), mL), respectively), yielding sub-fractions. Compound was 40:60 (100 mL), 0:100 (50 mL), respectively), yielding 18 sub-fractions. Compound 1 was obtained obtained from sub-fractions C7–C8 (104.7 mg, green oil) and compound 3 was obtained from from sub-fractions C7–C8 (104.7 mg, green and compound wasfrom obtained from sub-fractions sub-fractions C11–C12 (35.5 mg, yellow oil). oil) Sub-fraction A6 (4143mg) Column A was subjected C11–C12 (35.5 mg, yellow oil). Sub-fraction A6 (414 mg) from Column A was subjected to silica to a silica gel 60 (40–63 µm) column chromatography (Column D) and eluted with gradient asolvent gel 60 (40–63 µm) column chromatography (Column D) and eluted with gradient solvent systems of systems of n-hexane–acetone in increasing polarity (80:20 (500 mL), 70:30 (200 mL), 0:100 (50 mL), n-hexane–acetone in increasing polarity (80:20 (500 mL), 70:30 (200 mL), 0:100 mL),submitted respectively), respectively), yielding 25 sub-fractions. The sub-fractions D11–D13 (48.4 mg)(50 were to a yielding 25 sub-fractions. The sub-fractions D11–D13 (48.4 mg) were submitted to a Sephadex LH20 Sephadex LH20 column chromatography (Column E) and eluted with methanol yielding 16 subcolumn chromatography (Column E) and eluted with methanol 16 sub-fractions. Compound 2 fractions. Compound 2 was obtained from subfraction E7 (13.5yielding mg, yellow oil). All sub-fractions were was obtained subfraction E7 (13.5 oil). All sub-fractions were monitored by analytical monitored byfrom analytical TLC on silicamg, gel yellow 60 F254 aluminum sheets using n-hexane:acetone (6:4; v/v) TLC on silica gel 60detection F254 aluminum sheets using mobile detection as mobile phase; was performed at n-hexane:acetone daylight, 366 and(6:4; 254v/v) nm,asand afterphase; spraying with was performed atreagent daylight, 366 andby254 nm, and after spraying with reagent followed p-anisaldehyde followed heating. The fractionation and p-anisaldehyde isolation process is summarized by heating.4.The fractionation and isolation process is summarized in Figure 4. in Figure 3.4. Biological Activity Tests In vitro tests for activity against T. brucei rhodesiense (bloodstream trypomastigotes, strain STIB 900) and P. falciparum (intraerythrosctic forms, strains NF54 and K1) as well as for cytotoxicity against mammalian cells (L6 rat skeletal myoblasts) were performed according to established protocols as reported previously, e.g., in [18].
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3.4. Biological Activity Tests In vitro tests for activity against T. brucei rhodesiense (bloodstream trypomastigotes, strain STIB 900) and P. falciparum (intraerythrosctic forms, strains NF54 and K1) as well as for cytotoxicity against mammalian cells (L6 rat skeletal myoblasts) were performed according to established protocols as reported previously, e.g., in [18]. 3.5. Molecular Modeling and NMR Shift Calculations for Compound 3 All calculations were performed with molecular models of the dealkylated carboxamide part of 3, i.e., the isobutyl residue was omitted in order to save computation time. A molecular model of each, the 6S,9R-cis- and the 6R,9R-trans- diastereomer was generated with the software package MOE [19] and submitted to a low-mode molecular dynamics conformational search using the MMFF94x force field and standard settings. The energetically lowest conformer of each stereoisomer found in these searches was energy-minimized with Gaussian 03W [20] using the B3LYP density functional and the 6-31G++(2d,p) basis set. Subsequently, the NMR shielding tensors were computed with the same software and at the same level of theory using the GIAO formalism. A molecule of tetramethylsilane was treated in the same manner in order to obtain the reference value (34.9441 ppm). Chemical shift values were obtained by subtracting the TMS reference value from the values calculated for the protons mentioned in Table 1. 4. Conclusions The search for alkamides with anti-protozoal activity in the medicinal plant Acmella ciliata has resulted in the discovery of dioxacmellamide (3), a new alkamide with cyclic peroxide structure which shows considerable in vitro activity against P. falciparum with selectivity against the multiresistant K1 strain. It is very likely that this interesting compound is formed by 1,4-cycloaddition of oxygen to the diene moiety of the plant product spilanthol, and it could also be demonstrated that it can easily be obtained from the latter. This accessibility from the main constituent spilanthol will enable us to obtain larger quantities for in vivo tests and detailed studies of the mechanism of action of this new anti-plasmodial hit molecule. Such studies have been initiated. Supplementary Materials: MS and NMR spectra of 3 as well as details on the MS analyses are available online at: http://www.mdpi.com/1420-3049/21/6/765/s1. Acknowledgments: The authors thank CNPq and CAPES for the financial support and the scholarships. The authors also thank Professor Marcos Sobral for the identification of plant material and Associação dos Funcionários Fiscais do Estado de Santa Catarina (AFFESC) for providing the plant material. TJS gratefully acknowledges financial support from Apothekerstiftung Westfalen-Lippe. Parts of this study are an activity of MWB, TJS, LPS and RB within the Research Network Natural Products against Neglected Diseases (ResNet NPND, http://www.resnetnpnd.org/). Author Contributions: N.S. (doctorate student) contributed in collecting the plant material, phytochemical investigation, elucidation of compounds, data analyses and drafted the paper. J.S. (undergraduate student of the University of Münster) contributed in performing part of the laboratory work during a pharmacy practice internship at the UFSC laboratories. A.D.C.S. (doctorate student) performed some of the NMR measurements. A.B. contributed to NMR analyses and elucidation of compounds. L.P.S. contributed to L.C.M.S. analyses and elucidation of compounds. R.B. and M.K. performed the biological activity tests. M.W.B. supervised the laboratory work, data analyses and contributed to finalization of the manuscript. T.J.S. initiated the study, obtained and analyzed NMR spectra of compound 3, performed LC-MS analysis and finalized the manuscript together with M.W.B. Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–3 are available from the authors. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
